7454
Langmuir 2006, 22, 7454-7457
Influence of r-Hydroxylation of Glycolipids on Domain Formation in Lipid Monolayers Barbara Windschiegl and Claudia Steinem* Institut fu¨r Analytische Chemie, Chemo- und Biosensorik, UniVersita¨t Regensburg, 93040 Regensburg, Germany ReceiVed January 16, 2006. In Final Form: April 26, 2006 By means of fluorescence and scanning force microscopy (SFM), we investigated the phase behavior of lipid monolayers composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol (5/2/3) with either R-hydroxylated or nonhydroxylated galactocerebroside. Fluorescence images of lipid monolayers at the air-water interface demonstrate that, independent of the lipid mixture, phase separation occurs at low surface pressure up to 4-6 mN m-1, while an almost homogeneous phase is observed at larger surface pressures. However, by means of SFM of lipid monolayers transferred by the Langmuir-Blodgett technique at around 30 mN m-1, nanometer-sized domains became discernible in those lipid mixtures that contained galactocerebroside, while, in that without a glycolipid, no such domain formation was visible. Moreover, the R-hydroxy group of the galactocerebroside alters the size and the total area of the domains significantly.
Introduction A recent and important novel idea in the field of lipid membranes is the so-called raft hypothesis.1 The raft hypothesis proposes that naturally occurring lipids such as sphingomyelin (SM), cholesterol (Chol), and glycosphingolipids (GSLs) segregate in the plane of the membrane driven by distinct lipidlipid interactions.2,3 To support this hypothesis, domain formation in artificial membrane systems mimicking the raft composition has been investigated.4-14 Two different phases were observed: one is defined as the liquid disordered phase (ld), which is a liquid crystalline lamellar phase in which the lipids enjoy full freedom of diffusional, rotational, and conformational motions within the bilayer plane. The other one is defined as the liquid ordered (lo) phase, which is a liquid crystalline lamellar phase enriched in SM and Chol, in which the lipids diffuse freely along the plane of the bilayer, while keeping the fatty acyl chains in a predominately extended, ordered conformation.15 The localization of GSLs within the membrane is of particular interest. Most investigations were focused on the ganglioside GM1. In phase-separated two- and three-component systems, the * Corresponding author. Fax: (+49)
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(1) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572. (2) Brown, D. A.; London, E. J. Membr. Biol. 1998, 275, 17221-17224. (3) Silvius, J. R. Biochim. Biophys. Acta 2003, 1610, 174-183. (4) Yuan, C.; Furlong, J.; Burgos, P.; Johnston, L. J. Biophys. J. 2002, 82, 2526-2535. (5) Lawrence, J. C.; Saslowsky, D. E.; Edwardson, J. M.; Henderson, R. M. Biophys. J. 2003, 84, 1827-1832. (6) Giocondi, M. C.; Vie, V.; Lesniewska, E.; Goudonnet, J. P.; Le Grimellec, C. J. Struct. Biol. 2000, 131, 38-43. (7) Giocondi, M. C.; Milhiet, P. E.; Dosset, P.; Le Grimellec, C. Biophys. J. 2004, 86, 861-869. (8) Milhiet, P. E.; Vie, V.; Giocondi, M.-C.; Le Grimellec, C. Single Mol. 2001, 2, 109-112. (9) Milhiet, P. E.; Giocondi, M. C.; Le Grimellec, C. J. Biol. Chem. 2002, 277, 875-878. (10) Rinia, H. A.; Snel, M. M. E.; van der Eerden, J.; de Kruijff, B. FEBS Lett. 2001, 501, 92-96. (11) van Meer, G. Science 2002, 296, 855-857. (12) Wang, T. Y.; Silvius, J. R. Biophys. J. 2000, 79, 1478-1489. (13) Mesquita, R. M. R. S.; Melo, E.; Thompson, T. E.; Vaz, W. L. C. Biophys. J. 2000, 78, 3019-3025. (14) Milhiet, P. E.; Domec, C.; Giocondi, M. C.; Van Mau, N.; Heitz, F.; Grimellec, C. Biophys. J. 2001, 81, 547-555. (15) Lichtenberg, D.; Goni, F. M.; Heerklotz, H. Trends Biochem. Sci. 2005, 30, 430-436.
ganglioside GM1 preferentially clusters in the liquid condensed or liquid ordered phase.1,4,16-19 In biological membranes, several functions are ascribed to GSLs. They are important factors in cell signaling, growth, differentiation, cell adhesion, and antigenicity. Disregulation of signal transduction can cause various diseases, such as cancer or Alzheimer’s disease.20,21 In particular, it has been demonstrated that the composition of GSLs in tumors is considerably altered. For example, R-L-fucopyranosylceramide is upregulated in intestine cancer.22,23 Another observation is that R-hydroxylation of the fatty acid is unusually high in GSLs of human colonic cancer, human neuroblastoma,24 and chronic or acute lymphocytic leukemia.25 In neuroblastoma, one-fifth of the total number of ganglioside GM2 harbors an R-hydroxylated fatty acid, which changes its function significantly. The affinity of a monoclonal antibody to an R-hydroxylated d18:1-h16:0 asialo-GM2 was significantly larger than that to d18:1-16:0 asialo-GM2, the analogous nonhydroxylated GSL,25 which is discussed in terms of a different structural conformation leading to an enhanced exposure of the oligosaccharide on the cell surface. In healthy tissues, R-hydroxylated fatty acids are, however, also present in GSLs. GSLs containing hydroxy fatty acids are abundant in the skin, kidneys, stomach, and intestines.26 Ceramides, lipophilic intermediates of the GSL metabolism, are essential for the function of the skin containing unusually high concentrations of long fatty acids, which are partially hydroxylated at different positions.27 In myelin, galactocerebrosides are very (16) Hammond, A. T.; Heberle, F. A.; Baumgart, T.; Holowka, D.; Baird, B.; Feigenson, G. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6320-6325. (17) Vie, V.; Van Mau, N.; Lesnniewska, E.; Goudonnet, J. P.; Heitz, F.; LeGrimmelec, C. Langmuir 1998, 14, 4574-4583. (18) Yuan, C.; Johnston, L. J. Biophys. J. 2000, 79, 2768-2781. (19) Menke, M.; Ku¨nneke, S.; Janshoff, A. Eur. Biophys. J. 2002, 31, 317322. (20) Binder, W. H.; Barragan, V.; Menger, F. M. Angew. Chem., Int. Ed. 2003, 42, 5802-5827. (21) Zajchowski, L. D.; Robbins, S. M. Eur. J. Biochem. 2002, 269, 737-752. (22) Watanabe, K.; Matsubara, T.; Hakomori, S. J. Biol. Chem. 1976, 251, 2385-2387. (23) Lavie, Y.; Cao, H.; Bursten, S. L.; Giuliano, A. E.; Cabot, M. C. J. Biol. Chem. 1996, 271, 19530-19536. (24) Hakomori, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 225-232. (25) Ladisch, S.; Sweeley, C. C.; Becker, H.; Gage, D. J. Biol. Chem. 1989, 264, 12097-12105. (26) Eckhardt, M.; Yaghootfam, A.; Fewou, S. N.; Zoller, I.; Gieselmann, V. Biochem. J. 2005, 388, 245-254. (27) Kolter, T.; Sandhoff, K. Angew. Chem., Int. Ed. 1999, 38, 1532-1568.
10.1021/la060146x CCC: $33.50 © 2006 American Chemical Society Published on Web 08/02/2006
Letters
Langmuir, Vol. 22, No. 18, 2006 7455
Figure 2. Fluorescence micrographs of lipid monolayers at the air-water interface at 20 °C. POPC/SM/Chol (5/2/3) monolayer containing 10 mol % NFA-nGalCer at a surface pressure of (A1) 3 mN m-1 and (A2) 8 mN m-1. POPC/SM/Chol (5/2/3) monolayer containing 10 mol % HFA-nGalCer at a surface pressure of (B1) 3.5 mN m-1 and (B2) 8 mN m-1. All scale bars are 100 µm.
Figure 1. Chemical structure of POPC, SM, Chol, and galactocerebroside bearing a hydroxyl group in the R-position of the fatty acid (HFA-nGalCer) or not (NFA-nGalCer).
important, and 60-75% of these lipids contain R-hydroxylated fatty acids.28
Results and Discussion Here, we address the question of how a single hydroxy group in the R-position of a GSL influences the lateral organization of a lipid layer mimicking the outer leaflet of an eukaryotic membrane. We investigated by fluorescence and scanning force microscopy (SFM) artificial lipid monolayers composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), SM, and Chol (5/2/3) with either R-hydroxylated galactocerebroside (HFA-nGalCer) or nonhydroxylated galactocerebroside (NFA-nGalCer) (Figure 1) to visualize phase separation. The two different galactocerebrosides HFA-nGalCer and NFA-nGalCer were obtained from a commercially available mixture of both that was separated by thin-layer chromatography according to the procedure described by Radin.29 The success of separation was analyzed by mass spectrometry (for details, see Supporting Information). Different chain lengths and saturation degrees of the fatty acids are found in the HFA-nGalCer and NFA-nGalCer fraction, with the main fatty acid component of both lipids having a chain length of 24 C atoms (for details, see Experimental Section). Phase transitions of the galactocerebrosides of these compositions occur at around 80 °C.30-32 First, we used fluorescence microscopy at the air-water interface to characterize the phase behavior of mixed lipid monolayers with the aim to observe an influence of the R-hydroxy group on the phase behavior of the lipid mixtures. All measurements were performed on ultrapure water as the subphase at a temperature of 20 °C. Lipid monolayers were doped with
1 mol % β-BODIPY 500/510 C12-HPC. Fluorescence micrographs were taken at various surface pressures ranging from 0 to 32 mN m-1. Monolayers containing no or 10 mol % of NFAnGalCer or HFA-nGalCer in a mixture of POPC/SM/Chol (5/ 2/3) were investigated. In all lipid mixtures, dark round domains were monitored by fluorescence microscopy at low surface pressures (
